Fluorescent imaging techniques simultaneously capture different types of signals from multiple locations in a living cell.
Thousands of molecules in a single cell, such as proteins, ions, and other signaling molecules, work together to perform all kinds of functions. Nutrient absorption, memory storage և Differentiation of different tissues.
The interpretation of these molecules բոլոր all their interactions is a monumental task. Over the past 20 years, scientists have developed fluorescent journalists who can read the dynamics of individual molecules in cells. However, usually only one or two such signals can be seen at a time, as the microscope cannot distinguish the colors of daylight.
WITH: Researchers have now developed a method for imaging up to five different molecule types by measuring each signal from random, precise locations throughout the cell. This approach could allow scientists to learn much more about the complex signal networks that control most cell functions at MIT.
“There are thousands of molecules encoded by the genome, they interact in a way that we do not understand. “Only by looking at them at the same time can we understand their relationship,” said Boyden, a member of the McGovern Institute for Brain Research at MIT and the Koch Cancer Integrative Research Institute.
Researchers say that in a new study, Boyden and his colleagues used this technique to detect two populations of neurons that respond differently to calcium signals, which can affect the coding of long-term memories.
Boyden is the senior author of the study, which was published in 2020. On November 23, c Mobile:The main authors of the newspaper are MIT post-election doctor Changyang Linghu and post-graduate student Shannon John Onson.
To make molecular activity visible in a cell, scientists usually create reporters by merging the protein that the target molecule senses with the glowing protein. “This is how a smoke detector will feel the smoke and then release it,” said John Onson, a member of the Yan-Tan Molecular Therapy Center. The most commonly used gloss protein is the green fluorescent protein (GFP), which is based on a molecule originally found in daylight jellyfish.
“Usually a biologist can see one or two colors in a microscope at the same time. Many of the journalists there are green because they are based on the green fluorescent protein,” Boyden said. “What has been lacking so far is the ability to see more than one signal at a time.”
“Just like hearing the sound of one instrument from an orchestra is too far to fully appreciate a symphony,” says Lingu, “while adding multiple cell phone signals at the same time, our technology will help us understand the ‘symphony’ of ‘mobile activity.’
To be able to see the number of their signals, the researchers began to detect the signals by color, not location. They changed the existing correspondents, causing clusters to accumulate in their cells in different places. They did this by adding two small peptides to each reporter, which helped reporters form clear clusters in the cells.
“It’s like attaching a LEGO brick to reporter X and tying journalist Z to a piece of K’NEX. Only LEGO bricks will be attached to the other LEGO bricks, resulting in only X Reporter accumulating a larger mass of X Reporter, ”says John Onson. ,
With this technique, each cell appears with hundreds of clusters of fluorescent correspondents. After measuring the activity of each cluster under a microscope, based on changing luminescence, researchers can find out which molecule is being measured in each cluster by preserving the cell by dyeing peptide labels that are unique to each journalist. Peptide labels are invisible to the living cell, but they can be colored: seen after realizing a living image. This allows researchers to differentiate signals for different molecules, even though they are all the same color fluorescent in a living cell.
Using this approach, the researchers showed that they could see five different molecular signals in a single cell. To demonstrate the potential benefits of this strategy, they measured the activity of three molecules in parallel: calcium, cyclic AMP, and protein kinase A (PKA). These molecules form a signal network that is involved in various cellular functions in the body. In neurons, it plays an important role in transforming short-term input (from upstream neurons) into long-term changes, such as strengthening the connections between neurons. A process that is necessary to learn and to form new memories.
Using this imaging technique on pyramidal neurons in the hippocampus, researchers have discovered two new sublinguals with different calcium signaling dynamics. One population showed slow calcium responses. In other populations, neurons responded more rapidly to calcium. The latter population had higher PKA responses. Researchers believe that this high response may help maintain long-term changes in neurons.
Imaging signal networks
Researchers now plan to try this approach in living animals so that they can study how the signaling network responds to behavior and how it expands into other types of cells, such as immune cells. This technique can also be useful for comparing signal network patterns between healthy and diseased tissue cells.
In this article, the researchers showed that they can record five different molecular signals at once, and by changing their strategy, they believe they can reach 16. With more work, that number could reach hundreds, they say.
“It can really help solve some of these tough questions about how cell parts work together,” Boyden said. “One can imagine an era when we could watch everything that was going on in the living cell, or at least the part that was related to education, illness or treatment of disease.”
Read Real-Time Spy on Cellular Sound Symphony, which drives biology, for more information on this study.
Reference. “Spatial Multiplication of Fluorescent Reports for Dynamic Imaging of Signal Transmission Networks” by Changyang Linghu, Shannon L. Johnson, Pablo A. Valdes, Or A. Shemesh, Won Min Park, Demian Park, Kiryl D. Piatkevich, Asmamaw T. Vassi, Yikhi Liu, Boba An, Stephanie A. Barnes, Orhan T. Cheliker, Chun-Chen Yao, Chih-Chie (ay ey) Yu, Ru Wang, Katarzyna P. Adamala, Mark F. Beer, Amy E. Keating և Eduard S. Boyden, November 23, 2020 Mobile:,
DOI :: 10.1016 / j.cell.2020.10.035:
The study was funded by a McGovern Institute Friends Scholarship; Dou. Douglas Tan Scholarship; Lisa Young; Yang-tan Molecular Therapy Center; Do on Doer; open humanitarian project; HHMI-Simons Scholarship Program; Human Borders Science Program; US Army Research Laboratory; MIT Media Lab; Picower Institute Innovation Fund; National Institutes of Health, including the NIH Director Pioneer Award; ազգային National Science Foundation.